Weed control is a significant problem for farmers, and the use of herbicides to control weeds and other unwanted plants in cultivated fields has become a nearly universal practice. Broad-spectrum herbicides are particularly useful in combating wide varieties of weeds, but it is not always possible to identify a broad-spectrum herbicide that kills weeds without harming the crop plant. Production of hybrids or engineered crop varieties with specific herbicide resistance provides an attractive solution by allowing an herbicide to be used to kill weeds without significant damage to the resistant crop.
One class of broad-spectrum herbicides that is widely used and has proven particularly efficacious is the herbicidal sulfonamides. These compounds are well known in the art, and their general characteristics are discussed in U.S. Pat. No. 5,719,046. Herbicidal sulfonamides are effective at controlling both monocotyledonous and dicotyledenous weeds, while exhibiting low toxicity to mammals, birds, fish and other wild life. Sulfonamides also have short soil persistence, thereby reducing problems of toxic soil accumulation and run-off contamination.
Sulfonamides act as inhibitors of dihydropteroate synthase (DHPS), an enzyme of the folic acid synthesis pathway. The folic acid synthesis pathway is essential for the production of folic acid cofactors, which are required for the synthesis of purines, thymidylate, glycine, methionine, and several other compounds in all cells. Mammals have a carrier-mediated active transport system that allows mammalian cells to use pre-formed folic acid. In contrast, most microbes and plants lack this active transport system and therefore must synthesize folic acid de novo; they are therefore sensitive to sulfonamides.
Because sulfonamides act as competitive substrates of DHPS, mutations in the gene encoding this enzyme can occur that confer sulfonamide-insensitive DHPS activity. In bacteria, such naturally occurring resistance to sulfonamides is conferred by various bacterial R plasmids. The resistance genes (sul genes) found on R plasmids encode mutant versions of DHPS that are resistant to inhibition by sulfonamides (Guerineau, et al. (1990) Plasmid 23:35-41).
Naturally occurring resistance to herbicides in plants has not been extensively studied. However, two methods have been used to engineer such resistance: random mutagenesis and subsequent selection of resistant varieties; and specific genetic engineering and expression of resistant or insensitive proteins. Some researchers have selected for randomly occurring herbicide resistance through exposure of tissue culture cells to high herbicide levels. See, for instance, U.S. Pat. No. 5,718,079 (providing a method for growing maize with altered, herbicide resistant acetohydroxyacid synthase); and U.S. Pat. No. 4,757,011 (providing tobacco varieties resistant to herbicidal sulfonamides, where the resistance is induced through exposure of tissue cell cultures to inhibitory levels of sulfonamides). Others have mutagenized seed and subsequently selected for herbicide resistance (U.S. Pat. No. 5,084,082, providing soybean plants bearing at least one dominant random heritable mutation capable of conferring resistance to one or more herbicide).
The second method used to engineer resistance to herbicides involves transforming plants with specific nucleotide sequences encoding proteins that have herbicide resistant or insensitive enzyme activity. See, for instance, U.S. Pat. No. 5,605,011 (transformation of plants with sulfonylurea herbicide-insensitive acetolactate synthase, where the modified acetolactate synthase gene was isolated after exposure of cultured plant cells to high levels of a sulfonylurea herbicide).
Previous reports have shown that the sulI gene from bacterial R plasmid R46, when targeted into chloroplasts, confers sulfonamide resistance in plants (See U.S. Pat. Nos. 5,597,717 and 5,633,444). The present invention provides a method for targeting sulfonamide resistance genes to the mitochondrion and may provide optimal resistance.